An extensive flow cytometric evaluation continues of patients with autoimmune lymphoproliferative syndrome (ALPS) and their extended family members, on the basis of characterization of the expanded double-negative T-cell and B-cell populations. Double-negative T-cells have been demonstrated to be alpha beta TcR, CD57+, HLA-DR+, and CD45RA+. This study has been extended to characterize the double-negative T-cells more completely including B220 expression and gamma-delta TcR T-cells in all ALPS patients. In addition, we have expanded characterization of the B cells, directed at memory B cells using CD27 and B220 assessment in these patients and have documented a decrease in CD27+ B cells associated with altered repertoire of immunoglobuine heavy chain expression. These data are currently being assembled for publication. Functional studies of immunoregulatory T cells have failed in providing a consistent ex vivo indicator system for inhibition. But we have developed a quantitiative RTPCR assay for Fox P3 and have collected mRNA from T cells obtained from ALPS type Ia patients, family members and controls. These samples are now in the process of being evaluated for Fox P3 mRNA. In addition we have validated a flow cytometric assay for intracellular Fox P3 staining. This assay demonstrated that the bulk of Fox P3 expressing cells are CD4/CD25 T cells. Evaluation of the Fox P3 expressing cells in control subjects and ALPS patients suggests that Fox P3 expression is normal in ALPS despite the depression CD4/CD25 T cells. Finally, we have also extended the Fox P3 PCR and flow based assays to ATL cells based on the possibility that this disease represents a leukemic expansion of immunoregulatory T cells. The ATL studies show substantial differences between various ATL patients in terms of the level of FoxP3 expressed as measured by flow cytometry which has been corroborated by RTPCR testing. We are currently evaluating possible explanations for these differences including in vitro IL-2 independence